Figure 6. Proteasome gene expression is... | Rockefeller University Press

Figure 6.

$Proteasome gene expression is dependent on both PAX4 and α-PALNRF-1. (A) Strategy for generating α-PALNRF-1 and PAX4/α-PALNRF-1 double conditional KD mice. Following tamoxifen injections, removal of α-PALNRF-1 from one allele in the genome was verified using the U3/U5 primers (Table S3). (B and C) Mean body weights (g) (B) and percent survival (C) ± SEM of PAX4 KO, α-PALNRF-1 KD, PAX4/α-PALNRF-1 double conditional KD, and WT control mice are presented, following tamoxifen injections and muscle denervation. N = 10 mice per condition. **, P < 0.005 and ***, P < 0.0005 versus WT by one-tailed unpaired Student’s t test. (D) α-PALNRF-1 mRNA levels increase at 7–10 days after denervation, but not in denervated muscles from α-PALNRF-1 KD mice. Mice were injected with tamoxifen and their muscles were denervated (3, 7, or 10 days). RT-PCR using primers for α-PALNRF-1 was performed on mRNA preparations from innervated and denervated muscles. Means ± SEM are presented as a ratio to WT innervated. N = 4 mice per condition. *, P < 0.05 versus innervated in WT; #, P < 0.05 versus denervated (10 days). (E) α-PALNRF-1 accumulates in the nucleus at 7 days after denervation. Nuclear fractions from innervated and denervated (at 3, 7, and 10 days) muscles were analyzed by SDS-PAGE and immunoblotting. (F) Venn diagrams depicting the overlap between differentially expressed genes in muscles from α-PALNRF-1 KD and PAX4 KO mice following tamoxifen injections and muscle denervation. A significant overlap was detected for downregulated (P = 2.5e-29, Fisher Exact test) and upregulated (P = 3.3e-266, Fisher’s exact test) genes compared with denervated in WT. (G) Denervated muscles (10 days) were compared to innervated muscles by RNA sequencing. Presented are proteasome subunits whose induction was blocked in α-PALNRF-1 KD (yellow) and PAX4/α-PALNRF-1 double KD (green) mice, with the latter group of genes being fully contained within the first. (H) A heatmap representing the calculated fold changes for expression of proteasome genes in denervated (10 days) muscles from PAX4/α-PALNRF-1 double KD (left column), α-PALNRF-1 KD (middle column) and PAX4 KO (right column) mice versus innervated in WT. Asterisks denote significant fold changes (* Padj range 0.05–0.01, ** Padj 0.01–0.001, *** Padj < 0.001). (I) PAX4 and α-PALNRF-1 bind the promoter region of PSMB3 gene. ChIP was performed on denervated (10 days) muscles from WT mice using PAX4 or α-PALNRF-1 antibodies, or non-specific IgG control, and primers for PSMB3 gene. Data is plotted as mean fold change relative to IgG control ± SEM and represents three independent experiments. N = 3 mice per condition. *, P < 0.05 versus IgG by one-tailed unpaired Student’s t test. (J) The content of active assembled proteasomes increase in denervated (10 days) muscles, but not in muscles lacking both PAX4 and α-PALNRF-1. Measurement of proteasome content by native gels and immunoblotting, and proteasome peptidase activity by LLVY-AMC hydrolysis. Source data are available for this figure: SourceData F6.$

Proteasome gene expression is dependent on both PAX4 and α-PAL ^NRF-1 . (A) Strategy for generating α-PAL^NRF-1 and PAX4/α-PAL^NRF-1 double conditional KD mice. Following tamoxifen injections, removal of α-PAL^NRF-1 from one allele in the genome was verified using the U3/U5 primers (Table S3). (B and C) Mean body weights (g) (B) and percent survival (C) ± SEM of PAX4 KO, α-PAL^NRF-1 KD, PAX4/α-PAL^NRF-1 double conditional KD, and WT control mice are presented, following tamoxifen injections and muscle denervation. N = 10 mice per condition. **, P < 0.005 and ***, P < 0.0005 versus WT by one-tailed unpaired Student’s t test. (D) α-PAL^NRF-1 mRNA levels increase at 7–10 days after denervation, but not in denervated muscles from α-PAL^NRF-1 KD mice. Mice were injected with tamoxifen and their muscles were denervated (3, 7, or 10 days). RT-PCR using primers for α-PAL^NRF-1 was performed on mRNA preparations from innervated and denervated muscles. Means ± SEM are presented as a ratio to WT innervated. N = 4 mice per condition. *, P < 0.05 versus innervated in WT; #, P < 0.05 versus denervated (10 days). (E) α-PAL^NRF-1 accumulates in the nucleus at 7 days after denervation. Nuclear fractions from innervated and denervated (at 3, 7, and 10 days) muscles were analyzed by SDS-PAGE and immunoblotting. (F) Venn diagrams depicting the overlap between differentially expressed genes in muscles from α-PAL^NRF-1 KD and PAX4 KO mice following tamoxifen injections and muscle denervation. A significant overlap was detected for downregulated (P = 2.5e-29, Fisher Exact test) and upregulated (P = 3.3e-266, Fisher’s exact test) genes compared with denervated in WT. (G) Denervated muscles (10 days) were compared to innervated muscles by RNA sequencing. Presented are proteasome subunits whose induction was blocked in α-PAL^NRF-1 KD (yellow) and PAX4/α-PAL^NRF-1 double KD (green) mice, with the latter group of genes being fully contained within the first. (H) A heatmap representing the calculated fold changes for expression of proteasome genes in denervated (10 days) muscles from PAX4/α-PAL^NRF-1 double KD (left column), α-PAL^NRF-1 KD (middle column) and PAX4 KO (right column) mice versus innervated in WT. Asterisks denote significant fold changes (* Padj range 0.05–0.01, ** Padj 0.01–0.001, *** Padj < 0.001). (I) PAX4 and α-PAL^NRF-1 bind the promoter region of PSMB3 gene. ChIP was performed on denervated (10 days) muscles from WT mice using PAX4 or α-PAL^NRF-1 antibodies, or non-specific IgG control, and primers for PSMB3 gene. Data is plotted as mean fold change relative to IgG control ± SEM and represents three independent experiments. N = 3 mice per condition. *, P < 0.05 versus IgG by one-tailed unpaired Student’s t test. (J) The content of active assembled proteasomes increase in denervated (10 days) muscles, but not in muscles lacking both PAX4 and α-PAL^NRF-1. Measurement of proteasome content by native gels and immunoblotting, and proteasome peptidase activity by LLVY-AMC hydrolysis. Source data are available for this figure: SourceData F6.